Compositions and methods with efficacy against spores and other organisms

ABSTRACT

Compositions and methods for the disinfection of surfaces are provided. The compositions include at least about 40 weight percent of a C 1-6  alcohol, and a primary enhancer selected from protein denaturants. The disinfectant composition is characterized by a pH of less than about 3. Broad spectrum efficacy is achieved, and synergistic activity is exhibited against  C. difficile  spores.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/009,004, filed Jan. 28, 2016, which is a continuation of U.S. patentapplication Ser. No. 14/615,552, filed Feb. 6, 2015, which claims thebenefits of U.S. Provisional Patent Application Ser. No. 61/936,945,filed Feb. 7, 2014, and U.S. Provisional Patent Application Ser. No.62/067,068, filed Oct. 22, 2014, all of which are incorporated herein byreference.

TECHNICAL FIELD

Embodiments of the present invention provide compositions and methodshaving efficacy against spores such as Clostridium difficile spores, andother organisms. Acidified disinfectant compositions containing one ormore enhancers exhibit synergistic efficacy against spores such as C.difficile spores, and also exhibit excellent efficacy against fungi,bacteria, and viruses.

BACKGROUND OF THE INVENTION

Patients in healthcare facilities can sometimes contract seriousinfections. Such infections may be generally referred to ashealthcare-associated infections (HAIs). While most types of HAIs aredeclining, one infection, caused by the bacteria Clostridium difficile(C. difficile), remains at historically high levels. C. difficile is aspore-forming, Gram-positive anaerobic bacillus of the human intestineand is thought to be present in 2-5% of the adult population. PathogenicC. difficile strains produce multiple toxins, the mostwell-characterized of which are enterotoxin (Clostridium difficile toxinA) and cytotoxin (C. difficile toxin B), both of which can producediarrhea and inflammation in infected patients. The emergence of a new,highly toxic strain of C. difficile, resistant to fluoroquinoloneantibiotics, such as ciprofloxacin and levofloxacin have also beenreported. C. difficile infection causes diarrhea and other intestinalproblems and is linked to 14,000 deaths in the United States each year.

Control of C. difficile outbreaks present significant challenges tohealth care facilities. C. difficile spores survive routineenvironmental cleaning with detergents and hand hygiene withalcohol-based gels. The spores can survive on surfaces for long periodsof time. As a result, the bacteria can be cultured from almost anysurface. Once spores are ingested, their acid-resistance allows them topass through the stomach unscathed. They germinate and multiply intovegetative cells in the colon upon exposure to bile acids.

A variety of strategies have been proposed to kill C. difficile sporeson various surfaces, with limited success. Bleach-based compositionshave been employed for hard surfaces, and have been shown to reduce theenvironmental burden of C. difficile. but can be corrosive. Hydrogenperoxide-based compositions have also been proposed, includingcombinations of hydrogen peroxide and peracetic acid, a combination ofhydrogen peroxide and silver cation dry-mist system, and the so-calledAccelerated Hydrogen Peroxide (AHP). Peracids generally have poorstability and corrosive properties. Hydrogen peroxide is also prone todecomposition, and concentrated solutions can be highly corrosive.Alcohol-based sanitizers have not generally been effective. In fact,ethanol is sometimes used to store C. difficile spores.

A need remains for more stable, less corrosive compositions having goodefficiency against C. difficile spores.

Spores and other pathogenic infectious agents such as bacteria, fungi,viruses, fungal and bacterial spores, and conformationally alteredprions can be resistant to current sanitizers and cleansers. Chemicaland biological warfare agents can be fast-acting and pervasive. There isa continuing need for effective, easy to use products that will be safefor humans and the environment, that can decontaminate skin, andparticularly wounds, following chemical and/or biological warfare agentexposure, that can decontaminate surfaces to eliminate infectious agentssuch as conformationally altered prions, bacteria, fungi, viruses, andfungal and bacterial spores, and that can be used to decontaminatehomes, building materials, and furniture that has been infected withblack mold spores, and that can reduce the transmission of infectiouspathogens.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic view of a dispenser according to the presentinvention.

FIG. 2 is a graphical representation of log reduction (CFU/ml) againstC. difficile spores for test compositions as described herein.

FIG. 3 is a graphical representation of log reduction (CFU/ml) againstC. difficile spores for test compositions as described herein.

FIG. 4 is a graphical representation of log reduction (CFU/ml) againstC. difficile spores for test compositions as described herein.

FIG. 5 is a graphical representation of log reduction (CFU/ml) againstC. difficile spores for test compositions as described herein.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

In one or more embodiments, the present invention provides disinfectantcompositions. The physical form of the disinfectant composition is notparticularly limited, and in one or more embodiments, the compositionmay be presented as a liquid that is poured, pumped, sprayed, orotherwise dispensed, a gel, an aerosol, or a foam, including bothaerosol and non-aerosol foams. The disinfectant composition of thepresent invention may be employed on a wide variety of surfaces orsubstrates, including hard surfaces, soft surfaces, non-living(inanimate) surfaces, living tissue, skin, soil, porous, and non-poroussurfaces. For purposes of this specification, the term “surface” shouldbe understood to include skin. The compositions of the invention may beemployed to disinfect or otherwise sanitize inanimate objects such asinstruments, medical equipment, furniture, handrails, textiles, etc. Inone or more embodiments, the disinfectant composition may be presentedas a wipe, i.e. a tissue or cloth that is wiped over a surface.

The disinfectant compositions comprise at least one C₁₋₆ alcohol, i.e.an alcohol containing 1 to 6 carbon atoms. Such alcohols may be referredto as lower alkanols. In one or more embodiments, the disinfectantcompositions comprise at least one C₁₋₄ alcohol. Examples of loweralkanols include, but are not limited to, methanol, ethanol, propanol,butanol, pentanol, hexanol, and isomers and mixtures thereof. In one ormore embodiments, the alcohol comprises ethanol, propanol, or butanol,or isomers or mixtures thereof. In one or more embodiments, the alcoholcomprises isopropanol. In other embodiments, the alcohol comprisesethanol. In one or more embodiments, the disinfectant compositionscomprise a mixture of alcohols. In one or more embodiments, thedisinfectant compositions comprise a mixture of ethanol and isopropanol.In one or more embodiments, the disinfectant compositions comprisebutanol. In one or more embodiments, the disinfectant compositionscomprise a mixture of ethanol and butanol. In one or more embodiments,the disinfectant compositions comprise a mixture of n-butanol andisopropanol.

In one or more embodiments, the disinfectant composition comprises atleast about 40 percent by weight (wt. %) alcohol, based upon the totalweight of the disinfectant composition. In one or more embodiments, thedisinfectant composition comprises at least about 45 wt. %, in otherembodiments, at least about 50 wt. % alcohol, in other embodiments, atleast about 55 wt. %, in other embodiments, the disinfectant compositioncomprises at least about 60 wt. % alcohol, in other embodiments, thedisinfectant composition comprises at least about 65 wt. % alcohol, inyet other embodiments, the disinfectant composition comprises at leastabout 70 wt. % alcohol, and in still yet other embodiments, thedisinfectant composition comprises at least about 75 wt. % alcohol,based upon the total weight of disinfectant composition.

In one embodiment, the disinfectant composition comprises less thanabout 99 wt. % alcohol, in other embodiments, the disinfectantcomposition comprises less than about 98 wt. % alcohol, in otherembodiments, the disinfectant composition comprises less than about 95wt. % alcohol. In one embodiment, the disinfectant composition comprisesless than about 90 wt. % alcohol, in other embodiments, the disinfectantcomposition comprises less than about 85 wt. % alcohol, in otherembodiments, the disinfectant composition comprises less than about 80wt. % alcohol. More or less alcohol may be required in certaininstances, depending particularly on other ingredients and/or theamounts thereof employed in the composition.

In one or more embodiments, the disinfectant composition comprises fromabout 40 wt. % to about 99 wt. % alcohol, in other embodiments, fromabout 45 wt. % to about 98 wt. % alcohol, in other embodiments, thedisinfectant composition comprises from about 50 wt. % to about 95 wt. %of alcohol, based upon the total weight of the disinfectant composition.In one or more embodiments, the disinfectant composition comprises fromabout 40 wt. % to about 90 wt. % alcohol, in other embodiments, fromabout 45 wt. % to about 85 wt. % alcohol, in other embodiments, thedisinfectant composition comprises from about 50 wt. % to about 85 wt. %of alcohol, in other embodiments, from about 50 wt. % to about 85 wt. %,in yet other embodiments, the disinfectant composition comprises fromabout 60 wt. % to about 78 wt. % of alcohol, and in still otherembodiments, the disinfectant composition comprises from about 65 wt. %to about 75 wt. % of alcohol, based upon the total weight of thedisinfectant composition.

In any of the above embodiments, the disinfectant composition mayinclude one or more enhancers. Advantageously, the composition includesat least one primary enhancer that potentiates the efficacy of thedisinfectant composition. Examples of primary enhancers include proteindenaturants. Examples of primary enhancers include chaotropic agents.Examples of primary enhancers include amine-containing enhancers,α-aminoacids, salts of alkali metals, salts of alkaline earth metals,and anionic surfactants.

In any of the above embodiments, the disinfectant composition mayinclude an amine-containing enhancer. Examples of amine-containingenhancers include urea, thiourea, dimethyl urea, guanidine-HCl,guanidine thiocyanate, aminoguanidine bicarbonate, guanidine carbonate,guanidine phosphate, L-NG-nitroarginine, and aminoguanidine-HCL. In oneor more of the above embodiments, the enhancer comprises urea. In one ormore of the above embodiments, the enhancer comprises guanidine-HCl. Inone or more of the above embodiments, the enhancer comprisesaminoguanidine-HCl.

In any of the above embodiments, the disinfectant composition mayinclude an α-aminoacid. Examples of α-aminoacids includesulfur-containing aminoacids and nitro-containing aminoacids. Examplesof sulfur-containing aminoacids include L-cysteine and methionine.Examples of nitro-containing aminoacids include L-NG-nitroarginine.Examples of α-aminoacids include α-aminoacid chelators. Examples ofα-aminoacid chelators include the trisodium salt ofmethylglycinediacetic acid (Na₃MGDA). Na₃MGDA is commercially availableunder the trade name Trilon M from BASF.

Interestingly, not all chelators are primary enhancers. For example,acrylic acid/acrylamidomethyl propane sulfonic acid copolymer, which issold under the tradename CG4500 by Lubrizol Corporation, and is known tohave chelating ability, does not work as a primary enhancer. That is,acrylic acid/acrylamidomethyl propane sulfonic acid copolymer does notexhibit a synergistic enhancement of the efficacy of alcohol against C.difficile spores, even at a pH of less than 3. However, such chelatorsas acrylic acid/acrylamidomethyl propane sulfonic acid copolymer mayenhance the efficacy against C. difficile spores, when combined with aC1-6 alcohol and a primary enhancer at a pH of no more than 3.

In any of the above embodiments, the disinfectant composition mayinclude one or more salts of alkali metals and alkaline earth metals.Examples of salts include ammonium calcium, iron, lithium, magnesium,and sodium salts. Examples of salts include ammonium chloride, ammoniumiron citrate, calcium chloride, iron perchlorate, lithium perchlorate,lithium acetate, magnesium chloride, sodium chlorate, sodium chloride,sodium chlorite, and tris-HCl (tris is2-Amino-2-hydroxymethyl-propane-1,3-diol).

In any of the above embodiments, the disinfectant composition mayinclude one or more anionic surfactants. Anionic surfactants includesodium lauryl sulfate (SLS) (also known as sodium dodecyl sulfate (SDS))and sodium laureth sulfate (SLES).

Combinations of primary enhancers may also be employed. In one or moreembodiments, the disinfectant composition comprises Na₃MGDA and sodiumchloride. In one or more embodiments, the disinfectant compositioncomprises urea and sodium nitrite. In one or more embodiments, thedisinfectant composition comprises urea and SDS. In one or moreembodiments, the disinfectant composition comprises urea and SLES.

Advantageously, a synergistic sporicidal effect is observed when theenhancer is combined with alcohol at an acidic pH. In certainembodiments, enhancers that exhibit little or no efficacy on their ownagainst C. difficile spores provide an enhanced efficacy when combinedwith alcohol according to the present invention, and a further enhancedefficacy when the pH of the disinfectant composition is less than 3. Ithas surprisingly been found that, while disinfectant compositions showlittle or no efficacy against the spores, the combination of an enhancerand alcohol at a low pH exhibits synergistically enhanced efficacyagainst C. difficile spores.

The amount of enhancer is not particularly limited, so long as it is atleast an efficacy-enhancing amount. The minimum amount of enhancer thatcorresponds to an efficacy-enhancing amount can be determined bycomparing the log kill of spores achieved by a composition comprising analcohol to a composition comprising an alcohol and a given amount ofenhancer. The amount of enhancer below which no difference in log killis seen is an efficacy-enhancing amount. In other words, rapidsporicidal efficacy is observed at lower concentrations of alcohol whenan enhancer is present compared to when the enhancer is not present.

In one or more embodiments, the amount of primary enhancer is no morethan about 15 wt. %, in other embodiments, no more than about 10 wt. %,in other embodiments, no more than about 8 wt. %, in other embodiments,no more than about 5 wt. %, in other embodiments, no more than about 3wt. %, in other embodiments, no more than about 2 wt. %, in otherembodiments, no more than about 1 wt. %, in other embodiments, no morethan about 0.5 wt. %, based upon the total weight of the disinfectingcomposition.

In one or more embodiments, the amount of primary enhancer is greaterthan about 0.1 wt. %, in other embodiments, greater than about 0.5 wt.%, in other embodiments, greater than about 1 wt. %, in otherembodiments, greater than about 2 wt. %, in other embodiments, greaterthan about 3 wt. %, in other embodiments, greater than about 5 wt. %, inother embodiments, greater than about 8 wt. %, based upon the totalweight of the disinfecting composition.

In one embodiment, the enhancer is added in an amount of from about 0.1to about 20 wt. %, based upon the total weight of the disinfectantcomposition. In another embodiment, the amount of enhancer is from about0.25 to about 15 wt. %, and in yet another embodiment, from about 0.5 toabout 12 wt. %, based upon the total weight of the disinfectantcomposition. It will be understood that greater levels of enhancer canbe used, if desired, and are expected to perform equally as well. Itwill be understood that when a combination of primary enhancers isemployed, the amount of each primary enhancer may be reduced.

In one or more embodiments, the pH of the disinfectant composition isless than about 3, in other embodiments, less than or equal to about2.75, in other embodiments, less than or equal to about 2.5, in otherembodiments, less than or equal to about 2.3, in other embodiments, lessthan or equal to about 2, in other embodiments, the pH is 1.8 or less,in other embodiments, the pH is 1.6 or less, in other embodiments, thepH is 1.5 or less. In one or more embodiments, the pH of thedisinfectant composition is from about 0 to about 2.75. In one or moreembodiments, the pH of the disinfectant composition from about 0.1 toabout 2. In one or more embodiments, the pH of the disinfectantcomposition from about 0.5 to about 1.8. In one or more of the aboveembodiments, the pH of the disinfectant composition from about 1 toabout 1.5. The disinfectant composition may therefore be referred to asacidified, since the disinfectant composition has an acidic pH.

The disinfectant composition may be acidified by the addition of one ormore acids. The type of acid is not limited, however, weak acids are notpreferred. The acid should have a pKa of 5.4 (the pKa of citric acid) orless.

Examples of useful acidifying agents include mineral acids and organicacids. Mineral acids include, without limitation, hydrochloric acid,nitric acid, phosphoric acid, phosphoric acid, boric acid, and sulfuricacid. Organic acids include sulfonic acids, organophosphorus acids,carboxylic acids such as benzoic acids, propionic acids, phthalic acids,butyric acids, acetic acids, amino acids, and other substituted andunsubstituted organic acids.

Examples of organic acids include adipic acid, benzene 1,3,5tricarboxylic acid, chlorosuccinic acid, choline chloride, cis-aconiticacid, citramalic acid, citric acid, cyclobutane 1,1,3,3 tetracarboxylicacid, cyclohexane 1,2,4,5 tetracarboxylic acid, cyclopentane 1,2,3,4tetracarboxylic acid, diglycolic acid, fumaric acid, glutamic acid,glutaric acid, glyoxylic acid, isocitric acid, ketomalonic acid, lacticacid, maleic acid, malic acid, malonic acid, nitrilotriacetic acid,oxalacetic acid, oxalic acid, phytic acid, p-toluenesulfonic acid,salicylic acid, succinic acid, tartaric acid, tartronic acid,tetrahydrofuran 2,3,4,5 tetracarboxylic acid, tricarballylic acid,versene acids, 3-hydroxyglutaric acid, 2-hydroxypropane 1,3 dicarboxylicacid, glyceric acid, furan 2,5 dicarboxylic acid, 3,4-dihydroxyfuran-2,5dicarboxylic acid, 3,4-dihydroxytetrahydrofuran-2,5-dicarb oxylic acid,2-oxo-glutaric acid, dl-glyceric acid, and 2,5 furandicarboxylic acid.

It has been found that, in certain embodiments, acidifying thedisinfectant composition enhances the efficacy of the alcoholicsolutions against C. difficile.

In any of the above embodiments, the disinfectant composition mayinclude one or more secondary enhancers that, when a primary enhancer ispresent in combination with alcohol at a low pH, further enhance theefficacy of the composition. Surprisingly, the secondary enhancers donot significantly enhance the efficacy of the alcohol unless a primaryenhancer is also present. In one or more embodiments, the secondaryenhancer provides the benefit of maintaining good efficacy at a pHhigher than for compositions that do not include a secondary enhancer.

Examples of secondary enhancers include non-ionic surfactants, such asdecyl glucoside and polyalkoxylated dimethicones including PEG-12dimethicone. Examples of secondary enhancers also include organic acids,such as citric acid, lauric acid, tannic acid, ascorbic and iodoaceticacid. Examples of secondary enhancers include also include auxiliaryantimicrobial agents. Examples of secondary enhancers include alsoinclude oxidizing agents such as sodium nitrite. Examples of secondaryenhancers include sugars and sugar alcohols. Examples of secondaryenhancers include monosaccharides such as fructose. In one or moreembodiments, the secondary enhancer includes glycerol.

In one or more embodiments, the amount of secondary enhancer in thedisinfectant composition is zero. In one or more embodiments, the amountof secondary enhancer is no more than about 10 wt. %, in otherembodiments, no more than about 8 wt. %, in other embodiments, no morethan about 5 wt. %, in other embodiments, no more than about 3 wt. %, inother embodiments, no more than about 2 wt. %, in other embodiments, nomore than about 1 wt. %, in other embodiments, no more than about 0.5wt. %, based upon the total weight of the disinfecting composition.

In one or more embodiments, the amount of secondary enhancer is greaterthan about 0.1 wt. %, in other embodiments, greater than about 0.5 wt.%, in other embodiments, greater than about 1 wt. %, in otherembodiments, greater than about 2 wt. %, in other embodiments, greaterthan about 3 wt. %, in other embodiments, greater than about 5 wt. %, inother embodiments, greater than about 8 wt. %, based upon the totalweight of the disinfecting composition.

Examples of auxiliary antimicrobial agents include, but are not limitedto, triclosan, also known as 5-chloro-2(2,4-dichlorophenoxy) phenol(PCMX) and available from Ciba-Geigy Corporation under the tradenameIRGASAN®; chloroxylenol, also known as 4-chloro-3,5-xylenol, availablefrom Nipa Laboratories, Inc. under the tradenames NIPACIDE® MX or PX;hexetidine, also known as 5-amino-1,3-bis(2-ethylhexyl)-5-methyl-hexahydropyrimidine; chlorhexidine salts includingchlorhexidine gluconate and the salts ofN,N″-Bis(4-chlorophenyl)-3,12-diimino-2,4,11,14-tetraazatetradecanediimidiamide; 2-bromo-2-nitropropane-1; 3-diol, benzalkonium chloride;cetylpyridinium chloride; alkylbenzyldimethylammonium chlorides; iodine;phenol, bisphenol, diphenyl ether, phenol derivatives, povidone-iodineincluding polyvinylpyrrolidinone-iodine; parabens; hydantoins andderivatives thereof, including 2,4-imidazolidinedione and derivatives of2,4-imidazolidinedione as well as dimethylol-5,5-dimethylhydantoin (alsoknown as DMDM hydantoin or glydant); phenoxy ethanol; cis isomer of1-(3-chloroallyl)-3,5,6-triaza-1-azoniaadamantane chloride, also knownas quaternium-15 and available from Dow Chemical Company under thetradename DOWCIL™ 2000; diazolidinyl urea; benzethonium chloride;methylbenzethonium chloride; glyceryl laurate, transition metalcompounds such as silver, copper, magnesium, zinc compounds, hydrogenperoxide, chlorine dioxide, anilides, bisguanidines, tropolone,C₆₋₁₀-alkane diols such as hexanediol, octanediol, and decanediol, andmixtures thereof. In one or more embodiments, the about of auxiliaryantimicrobial agent is from about 0.1 to about 1 wt. %, based upon thetotal weight of the disinfectant composition. In one or moreembodiments, the auxiliary antimicrobial agent is selected frombenzalkonium chloride and chlorhexidine gluconate.

The composition can further comprise a wide range of optionalingredients, with the proviso that they do not deleteriously affect thesanitizing efficacy of the composition. By deleterious is meant that thedecrease in the log reduction is not de minimus, or in other words, thelog reduction of C. difficile spores does not decrease by more thanabout 0.5. The CTFA International Cosmetic Ingredient Dictionary andHandbook, Eleventh Edition 2005, and the 2004 CTFA International Buyer'sGuide, both of which are incorporated by reference herein in theirentirety, describe a wide variety of non-limiting cosmetic andpharmaceutical ingredients commonly used in the skin care industry, thatare suitable for use in the compositions of the present invention.Nonlimiting examples of functional classes of ingredients are describedat page 537 of this reference. Examples of these functional classesinclude: abrasives, anti-acne agents, anticaking agents, antioxidants,binders, biological additives, bulking agents, chelating agents,chemical additives; colorants, cosmetic astringents, cosmetic biocides,denaturants, drug astringents, emulsifiers, external analgesics, filmformers, foam surfactants, fragrance components, humectants, opacifyingagents, plasticizers, preservatives (sometimes referred to asantimicrobials), propellants, reducing agents, skin bleaching agents,skin-conditioning agents (emollient, miscellaneous, and occlusive), skinprotectants, solvents, surfactants, foam boosters, hydrotropes,solubilizing agents, suspending agents (nonsurfactant), sunscreenagents, ultraviolet light absorbers, detackifiers, and viscosityincreasing agents (aqueous and nonaqueous). Examples of other functionalclasses of materials useful herein that are well known to one ofordinary skill in the art include solubilizing agents, sequestrants,keratolytics, topical active ingredients, and the like.

It has been discovered that the combination of alcohol and enhancer atlow pH exhibits enhanced antimicrobial efficacy. Advantageously,auxiliary antimicrobials, some of which can be harsh on skin, are notrequired. In certain embodiments, the disinfectant composition does notcontain any auxiliary antimicrobial ingredients. Any antimicrobialingredient other than the combination of alcohol, enhancer and acid maybe referred to as an auxiliary antimicrobial agent. In one or moreembodiments, the amount of auxiliary antimicrobial agent (includingpreservatives) is less than about 1 wt. %, in other embodiments, lessthan about 0.5 wt. %, in other embodiments, less than about 0.25 wt. %,based upon the total weight of the disinfectant composition. In one ormore embodiments, the amount of auxiliary antimicrobial agent (includingpreservatives) is less than about 0.1 wt. %, in other embodiments, lessthan about 0.05 wt. %, in other embodiments, less than about 0.01 wt. %,based upon the total weight of the disinfectant composition. In anotherembodiment, the disinfectant composition is devoid of auxiliaryantimicrobial agents.

Advantageously, certain ingredients that have been designated ascritical to current sporicidal compositions can be limited in thedisinfectant composition of the present invention. For example,hypochlorous acid and precursors thereof are not necessary, and can belimited, if desired, to less than about 0.5 wt. %, or in anotherembodiment to less than about 0.1 wt. %, based upon the total weight ofthe disinfectant composition. In another embodiment, the disinfectantcomposition is devoid of hypochlorous acid.

In one or more embodiments, the amount of peroxyacids such as peraceticacid may be limited. When limited, in one or more embodiments, theamount of peroxyacid may be less than 0.125 wt. %, in other embodimentsless than about 0.08 wt. %, based upon the total weight of thedisinfectant composition. In another embodiment, the disinfectantcomposition is devoid of peroxyacid.

In one or more embodiments, the amount of peroxide may be limited, ifdesired, to less than about 0.5 wt. %, or other embodiments to less thanabout 0.1 wt. %, based upon the total weight of the disinfectantcomposition. In another embodiment, the disinfectant composition isdevoid of peroxide.

Indeed, any component other than the alcohol, enhancer, acidifier, andoptionally a secondary enhancer, is not necessary to achieveantimicrobial efficacy and can optionally be limited to less than about0.5 wt. %, if desired to less than about 0.1 wt. %, if desired to lessthan about 0.01 wt. %, or if desired to less than about 0.001 wt. %. Itwill be understood that the balance of the disinfectant composition may,in certain embodiments, include water or other suitable solvent. In oneembodiment, the disinfectant composition is devoid of any componentother than alcohol, enhancer, acidifier, and optionally water or othersuitable solvent.

The disinfectant composition may be prepared by simply mixing thecomponents together. In one embodiment, where one or more components isobtained as a solid powder, the disinfectant composition may be preparedby a method comprising dispersing the solid powder in water or alcoholwith slow to moderate agitation, and then adding other ingredients asdesired, and mixing until the mixture is homogeneous.

In one embodiment, where the disinfectant composition is in liquid form,the percent solids of the disinfectant composition is less than about 6percent, in another embodiment, less than about 5 percent, in yetanother embodiment, less than about 4 percent, in still anotherembodiment, less than about 3 percent, in another embodiment, less thanabout 2 percent, in yet another embodiment, less than about 1 percent.The percent solids can be determined by various methods known in theart.

Advantageously, it has been found that compositions according to thepresent invention have efficacy against a broad spectrum of grampositive and gram negative bacteria, fungi, parasites, fungal andbacterial spores, enveloped and non-enveloped viruses, and prions (CJD,CWD, B SE, Scrapie). One or more embodiments of the present inventionexhibit efficacy against one or more of spores of Bacillus anthracis,Bacillus cereus, Clostridium difficile, Clostridium botulinum, andClostridium tetani.

Unexpectedly, when an enhancer is combined with alcohol at a low pH,according to the present invention, sporicidal activity is enhanced,i.e. potentiated. In one or more embodiments, the disinfectantcomposition is effective at killing C. difficile spores. In one or moreembodiments, the disinfectant composition is also effective in killinggram negative and gram positive bacteria, fungi, parasites,non-enveloped and enveloped viruses. In one or more embodiments, thedisinfectant composition has rapid antimicrobial efficacy againstbacteria such as Staphylococcus aureus, methicillin-resistant S. aureus,Escherichia coli, Pseudomonas aeruginosa, Serratia marcescens, fungisuch as Candida albicans and Aspergillus niger, and black mold sporesStachybotrys chartanim. In one or more embodiments, the disinfectantcomposition has rapid efficacy against skin microflora, includingresident and transient skin microflora.

Thus, the present invention further provides a method for killing orinactivating microbes such as C. difficile spores on a surfacecomprising applying, to the surface, an effective amount of adisinfectant composition as described herein. The disinfectantcomposition may be employed on a wide variety of surfaces or substrates,including hard surfaces, soft surfaces, skin, non-skin, animate,inanimate, porous, and non-porous surfaces.

Unless otherwise specified, the term log reduction refers to log₁₀reduction. In one or more embodiments, the method provides a logreduction against spores of at least about 1 in less than about 1minute. In other embodiments, the method provides a log reductionagainst spores of at least about 1.5 in less than about 1 minute. In yetother embodiments, the method provides a log reduction against spores ofat least about 2 in less than about 1 minute. In other embodiments, themethod provides a log reduction against spores of at least about 2.5 inless than about 1 minute. In yet other embodiments, the method providesa log reduction against spores of at least about 3 in less than about 1minute.

In one or more embodiments, the method provides a log reduction againstspores of at least about 1 in less than about 30 seconds. In otherembodiments, the method provides a log reduction against spores of atleast about 1.5 in less than about 30 seconds. In yet other embodiments,the method provides a log reduction against spores of at least about 2in less than about 30 seconds. In other embodiments, the method providesa log reduction against spores of at least about 2.5 in less than about30 seconds. In yet other embodiments, the method provides a logreduction against spores of at least about 3 in less than about 30seconds.

In one or more embodiments, the method provides a log reduction againstspores on inanimate, hard, non-porous surfaces of at least about 6 in 10minutes or less. In other embodiments, the method provides a logreduction against spores of at least about 6 in about 7 minutes or less.In yet other embodiments, the method provides a log reduction againstspores of at least about 6 in about 6 minutes or less. In otherembodiments, the method provides a log reduction against C. difficilespores on inanimate, hard, non-porous surfaces of at least about 6 inabout 10 minutes or less. In yet other embodiments, the method providesa log reduction against C. difficile spores on inanimate, hard,non-porous surfaces of at least about 6 in about 7 minutes or less. Inyet other embodiments, the method provides a log reduction against C.difficile spores on inanimate, hard, non-porous surfaces of at leastabout 6 in about 6 minutes or less.

In one or more embodiments, the method provides a log reduction againstC. difficile spores of at least about 1 in less than about 1 minute. Inother embodiments, the method provides a log reduction against C.difficile spores of at least about 1.5 in less than about 1 minute. Inyet other embodiments, the method provides a log reduction against C.difficile spores of at least about 2 in less than about 1 minute. Inother embodiments, the method provides a log reduction against C.difficile spores of at least about 2.5 in less than about 1 minute. Inyet other embodiments, the method provides a log reduction against C.difficile spores of at least about 3 in less than about 1 minute.

In one or more embodiments, the method provides a log reduction againstC. difficile spores of at least about 1 in less than about 30 seconds.In other embodiments, the method provides a log reduction against C.difficile spores of at least about 1.5 in less than about 30 seconds. Inyet other embodiments, the method provides a log reduction against C.Difficile spores of at least about 2 in less than about 30 seconds. Inother embodiments, the method provides a log reduction against C.difficile spores of at least about 2.5 in less than about 30 seconds. Inyet other embodiments, the method provides a log reduction against C.difficile spores of at least about 3 in less than about 30 seconds.

In one or more embodiments, the method provides a log reduction againstC. difficile spores on skin of at least about 1 in less than about 1minute. In other embodiments, the method provides a log reductionagainst C. difficile spores on skin of at least about 1.5 in less thanabout 1 minute. In yet other embodiments, the method provides a logreduction of C. difficile spores on skin of at least about 2 in lessthan about 1 minute. In other embodiments, the method provides a logreduction against C. difficile spores on skin of at least about 2.5 inless than about 1 minute. In yet other embodiments, the method providesa log reduction against C. difficile spores on skin of at least about 3in less than about 1 minute.

In one or more embodiments, the method provides a log reduction againstC. difficile spores on skin of at least about 1 in less than about 30seconds. In other embodiments, the method provides a log reductionagainst C. difficile spores on skin of at least about 1.5 in less thanabout 30 seconds. In yet other embodiments, the method provides a logreduction against C. difficile spores on skin of at least about 2 inless than about 30 seconds. In other embodiments, the method provides alog reduction against C. difficile spores on skin of at least about 2.5in less than about 30 seconds. In yet other embodiments, the methodprovides a log reduction against C. difficile spores on skin of at leastabout 3 in less than about 30 seconds.

The methods of the present invention include the step of applying andisinfectant composition to a surface.

Advantageously, good efficacy is achieved by the methods of the presentinvention when the disinfectant composition is applied to the surface atstandard temperature and at close to standard pressure. In one or moreembodiments, the temperature of the disinfectant composition whenapplied to the surface may be less than about 150° F., in otherembodiments, less than about 120° F., and in other embodiments, lessthan about 105° F. In one or more embodiments, the temperature of thedisinfectant composition may be in the range of from about 40° F. toabout 150° F. in other embodiments in the range of from about 40° F. toabout 105° F., and in other embodiments, in the range of about 70° F. to105° F.

Although the liquid disinfectant compositions of the present inventionmay be applied to the surface to be cleaned by spraying, no highpressure application is required. During this step, the disinfectantcomposition may be brought into contact with the target surface inbursts or in a continuous manner by circulating, flooding, spraying,foaming or fogging. The step may also be carried out by forming a twophase annular mist of antimicrobial treatment solution and air.

Advantageously, the methods of the present invention provide goodefficacy against spores within 10 minutes or less. Embodiments of theinvention provide good efficacy against spores within 7 minutes or less.Embodiments of the invention provide good efficacy against spores within6 minutes or less. Embodiments of the invention provide good efficacyagainst spores within 5 minutes or less. Embodiments of the inventionprovide good efficacy against spores within 2 minutes or less.Embodiments of the invention provide good efficacy against spores within1 minute or less. Embodiments of the invention provide good efficacyagainst spores within 30 seconds or less. In any of the embodimentsdisclosed herein, the spores may include C. difficile spores.Advantageously, the methods of the present invention provide goodefficacy against C. difficile spores within 5 minutes or less.Embodiments of the invention provide good efficacy against C. difficilespores within 2 minutes or less. Embodiments of the invention providegood efficacy against C. difficile spores within 1 minute or less.Embodiments of the invention provide good efficacy against C. difficilespores within 30 seconds or less. Thus, in one or more embodiments, theduration of contact of the disinfectant composition with the targetsurface is from about 20 seconds to 5 minutes, in other embodiments,from about 25 seconds to about 2 minutes, and in other embodiments, fromabout 30 seconds to about 1 minute. It will be understood that, in someembodiments, a longer contact time is advantageous, and in one or moreembodiments, the contact time may be up to 30 minutes, and in otherembodiments, up to about 60 minutes.

The amount of disinfectant composition to be applied to the targetsurface is not particularly limited. At a minimum, a sufficient amountof disinfectant composition should be applied to substantially wet thesurface such that the surface will remain wet for the desired contacttime, noting that there will be some evaporation of the disinfectantcomposition.

Any amount of the disinfectant composition may be used for eachapplication, so long as it is at least an effective amount to contactsubstantially the entire target surface and keep it wet for a contacttime of at least 30 to 60 seconds. In one or more embodiments, theamount of the disinfectant composition is sufficient to contactsubstantially the entire target surface and keep it wet for a contacttime of at least 5 minutes. In one or more embodiments, the amount ofthe disinfectant composition is sufficient to contact substantially theentire target surface and keep it wet for a contact time of at least 6minutes. In one or more embodiments, the amount of the disinfectantcomposition is sufficient to contact substantially the entire targetsurface and keep it wet for a contact time of at least 7 minutes. In oneor more embodiments, the amount of the disinfectant composition issufficient to contact substantially the entire target surface and keepit wet for a contact time of at least 10 minutes. In one or moreembodiments, the amount of the disinfectant composition is sufficient tocontact substantially the entire target surface and keep it wet for acontact time of at least 30 minutes. In one or more embodiments, theamount of the disinfectant composition is sufficient to contactsubstantially the entire target surface and keep it wet for at least 60minutes.

In one or more embodiments, the sporicidal disinfectant composition maybe prepared by combining two or more liquid pre-mix compositions. Afirst pre-mix composition may comprise a concentrate of the primaryenhancer, and a second pre-mix composition may comprise a concentrate ofthe alcohol, such that combination of the pre-mix compositions resultsin an disinfectant composition comprising alcohol and a primary enhanceras described hereinabove.

In other embodiments, a first pre-mix composition may comprise aconcentrate of the alcohol and primary enhancer, and the second pre-mixcomposition may comprise a diluent, such that combination of the pre-mixcompositions results in an disinfectant composition comprising alcoholand a primary enhancer at the concentrations as described hereinabove.

The pre-mix components may be dispensed from physically separatepackages or from a unitary package having non-communicating chambers.For purposes of this specification, the term dual dispenser apparatusrefers to a configuration where multiple liquid components are dispensedfrom a plurality of physically separate packages, and also refers to aconfiguration where multiple liquid components are dispensed from aunitary package having a plurality of non-communicating chambers, eachchamber having an orifice through which an aliquot of a component isdispensed.

In one or more embodiments, aliquots of the pre-mix components aredispensed substantially simultaneously, such that the liquid aliquotsare commingled. In particular embodiments, the aliquots are dispensedthrough orifices that are configured to enable the commingling of thealiquots. It will be understood that the dispenser may take a variety offorms, and may include a variety of components and configurations inorder to cause the desired comingling of aliquots of the pre-mixcomponents and dispensing of a product.

One embodiment of an exemplary dispenser is shown in FIG. 1 and isgenerally indicated by the numeral 100. Dispenser 100 may include afirst reservoir 102 containing a first liquid pre-mix component (e.g.concentrated primary enhancer pre-mix component), and a second reservoir104 containing a second liquid pre-mix component (e.g. alcoholic diluentpre-mix component). The pH-adjusting agent may be present in either orboth of the pre-mix components. As will be apparent to those skilled inthe art, and as indicated above, the first and second reservoirs 102 and104 are not in direct communication with one another, and the first andsecond pre-mix components are therefore stored separately within thedispenser. Although separate reservoirs are shown in FIG. 1, it iscontemplated that the first and second reservoirs 102 and 104 may beprovided as physically separate chambers in a single package. Each ofthe first and second reservoirs 102 and 104 is impervious to fluidtransfer therethrough, except through inlet passages 106 and 108,respectively.

In one or more embodiments, the present invention provides a method ofpreparing an disinfectant composition, the method comprising the stepsof providing a dispenser having a first reservoir containing a firstliquid pre-mix, and a second reservoir containing a second liquidpremix, wherein the dispenser is adapted to dispense an aliquot of thefirst pre-mix and an aliquot of the second pre-mix, such that thealiquots commingle. Upon commingling, the aliquots of the first pre-mixand second pre-mix form an disinfectant composition comprising analcohol and a primary enhancer at a pH of less than about 3.

In certain embodiments, a first pump 110 may be in fluid communicationwith the first reservoir 102 through the inlet passage 106, and a secondpump 112 may be in fluid communication with the second reservoir 104through the inlet passage 108. First and second pumps 110 and 112 may beany type of pump known to those skilled in the art and suitable forconveying the first and second liquid pre-mix components from the firstand second reservoirs 102 and 104. In one or more embodiments, the pumps110 and 112 may both be positive displacement pumps. The first andsecond pumps 110 and 112 discharge the first and second pre-mixcomponents through outlet passages 114 and 116, respectively. In certainembodiments, the output or displacement of the first and second pumps110 and 112 may be adjustable to vary the rate of fluid flowtherethrough. While the exemplary dispenser 100 shown and describedincludes first and second pumps 110 and 112, it is also contemplatedthat a single pump may be used, and may be in fluid communication withboth the first and second reservoirs 102 and 104.

The outlet passages 114 and 116 may each extend to a mixing nozzle 118where the first and second pre-mix components are comingled to form andisinfectant composition. The features and dimensions of the mixingnozzle 118 may be adjusted to vary the volume of each pre-mix aliquot,as well as the rate of mixing and comingling of the first and secondpre-mix components. The mixing nozzle 118 includes a dispensing passage120 through which the disinfectant composition is dispensed.

In certain embodiments, the first and second pumps 110 and 112 may beadjusted to produce substantially the same flow rate of the first andsecond pre-mix components therethrough. In other embodiments, the pumps110 and 112 may be adjusted to provide different flow rates, and incertain embodiments, the pre-mix components may be dispensedsequentially.

In certain embodiments, the first and second pumps 110 and 112 may beadjusted to select substantially the same aliquot volume for the firstand second pre-mix components. In other embodiments, the pumps 110 and112 may be adjusted to provide different aliquot volumes.

In one or more embodiments, the first and second pumps 110 and 112 maybe adapted to dispense a single dose of composition upon actuation. Inthe same or other embodiments, the first and second pumps 110 and 112may be adapted to produce a continuous flow of the disinfectantcomposition upon actuation.

In one or more embodiments, the first pre-mix may include a concentratedform of the compositions of the present invention, and the secondpre-mix may include a diluent, such that upon being dispensed, thecombination forms a composition that includes the amounts of componentstaught herein.

Advantageously, embodiments of the present invention provide easy to useproducts that are safe for humans and the environment, and that candecontaminate skin, and particularly wounds. Following chemical and/orbiological warfare agent exposure, embodiments of the present inventioncan contain and/or destroy the agent, preventing cutaneous penetrationand further contamination. Embodiments of the present invention candecontaminate surfaces to eliminate infectious agents such asconformationally altered prions, bacteria, fungi, viruses, and fungaland bacterial spores, and that can be used to decontaminate homes,building materials, and furniture that has been infected with black moldspores. Embodiments of the present invention can reduce the transmissionof infectious pathogens.

In order to demonstrate the practice of the present invention, thefollowing examples have been prepared and tested. The examples shouldnot, however, be viewed as limiting the scope of the invention. Theclaims will serve to define the invention.

EXAMPLES

Example 1 was a solution of 70 wt. % ethanol in water, to which 12 Nhydrochloric acid had been added to achieve a pH of about 1.5.

Example 2 was a solution of 5 wt. % urea in water, to which 12 Nhydrochloric acid had been added to achieve a pH of about 1.5.

Example 3 was a solution of 70 wt. % ethanol and 5 wt. % urea in water,to which 12 N hydrochloric acid had been added to achieve a pH of about1.5.

Example 4 was a solution of 70 wt. % ethanol in water, to which 12 Nhydrochloric acid had been added to achieve a pH of about 5.

Example 5 was a solution of 5 wt. % urea in water, to which 12 Nhydrochloric acid had been added to achieve a pH of about 5.

Example 6 was a solution of 70 wt. % ethanol and 5 wt. % urea in water,to which 12 N hydrochloric acid had been added to achieve a pH of about5.

The samples were tested for efficacy against C. difficile spores,according to the ASTM E2783-11: “Standard Test Method for Assessment ofAntimicrobial Activity for Water Miscible Compounds Using a Time-KillProcedure.”

Contact time was 30 seconds. Temperature was room temperature. Resultsare shown in Table 1 and FIG. 2.

TABLE 1 Log₁₀ Reduction Example Composition pH C. difficile spores 1 70%ethanol 1.5 0.51 2 5% urea 1.5 0.17 3 70% ethanol + 5% urea 1.5 2.78 470% ethanol 5.0 −0.35 5 5% urea 5.0 0.07 6 70% ethanol + 5% urea 5.0−0.49

Similar results were achieved when samples were prepared and tested asdescribed above, but varying the type of acid that was used to adjustthe pH. Nitric acid, sulfuric acid, hydrochloric acid, hydrobromic acid,hydroiodic acid, and perchloric acid were tried instead of hydrochloricacid, and all gave similar results.

Example 7 was a solution of 70 wt. % ethanol in water, to which 12 Nhydrochloric acid had been added to achieve a pH of about 1.5.

Example 8 was a solution of 5 wt. % aminoguanidine-HCl in water, towhich 12 N hydrochloric acid had been added to achieve a pH of about1.5.

Example 9 was a solution of 70 wt. % ethanol and 2 wt. %aminoguanidine-HCl in water, to which 12 N hydrochloric acid had beenadded to achieve a pH of about 1.5.

Example 10 was a solution of 70 wt. % ethanol and 5 wt. %aminoguanidine-HCl in water, to which 12 N hydrochloric acid had beenadded to achieve a pH of about 2.

Example 11 was a solution of 70 wt. % ethanol in water, to which 12 Nhydrochloric acid had been added to achieve a pH of about 5.

Example 12 was a solution of 5 wt. % aminoguanidine-HCl in water, towhich 12 N hydrochloric acid had been added to achieve a pH of about 5.

Example 13 was a solution of 70 wt. % ethanol and 5 wt. %aminoguanidine-HCl in water, to which 12 N hydrochloric acid had beenadded to achieve a pH of about 5.

As for Examples 1-4, samples 7-13 were tested for efficacy against C.difficile spores, according to the ASTM E2783-11: “Standard Test Methodfor Assessment of Antimicrobial Activity for Water Miscible CompoundsUsing a Time-Kill Procedure.” Contact time was 30 seconds. Temperaturewas room temperature. Results are shown in Table 2 and FIG. 3.

TABLE 2 Log₁₀ Reduction Example Composition pH C. difficile spores 7 70%ethanol 1.5 0.51 8 5% aminoguanidine-HC1 1.5 0.32 9 70% ethanol + 2% 1.52.63 aminoguanidine-HC1 10 70% ethanol + 5% 1.5 >2.93 aminoguanidine-HC111 70% ethanol 5.0 −0.35 12 5% aminoguanidine-HC1 5.0 0.20 13 70%ethanol + 5% 5.0 0.24 aminoguanidine-HC1

Example 14 was a solution of 80 wt. % ethanol in water, to which 12 Nhydrochloric acid had been added to achieve a pH of about 1.5.

Example 15 was a solution of 80 wt. % ethanol and 2 wt. %aminoguanidine-HCl in water, to which 12 N hydrochloric acid had beenadded to achieve a pH of about 1.5.

Example 16 was a solution of 80 wt. % ethanol, 2.5 wt. %aminoguanidine-HCl and 2.5 wt % urea in water, to which 12 Nhydrochloric acid had been added to achieve a pH of about 1.5.

Example 17 was a solution of 90 wt. % ethanol in water, to which 12 Nhydrochloric acid had been added to achieve a pH of about 1.5.

Example 18 was a solution of 90 wt. % ethanol, and 2 wt %aminoguanidine-HCl in water, to which 12 N hydrochloric acid had beenadded to achieve a pH of about 1.5.

Example 19 was a solution of 90 wt. % ethanol, 2.5 wt. %aminoguanidine-HCl and 2.5 wt % urea in water, to which 12 Nhydrochloric acid had been added to achieve a pH of about 1.5.

Example 20 was a solution of 80 wt. % ethanol in water, to which 12 Nhydrochloric acid had been added to achieve a pH of about 5.

Example 21 was a solution of 80 wt. % ethanol and 2 wt. %aminoguanidine-HCl in water, to which 12 N hydrochloric acid had beenadded to achieve a pH of about 5.

Example 22 was a solution of 80 wt. % ethanol, 2.5 wt. %aminoguanidine-HCl and 2.5 wt % urea in water, to which 12 Nhydrochloric acid had been added to achieve a pH of about 5.

Example 23 was a solution of 90 wt. % ethanol in water, to which 12 Nhydrochloric acid had been added to achieve a pH of about 5.

Example 24 was a solution of 90 wt. % ethanol and 2 wt. %aminoguanidine-HCl in water, to which 12 N hydrochloric acid had beenadded to achieve a pH of about 5.

Example 25 was a solution of 90 wt. % ethanol, 2.5 wt. %aminoguanidine-HCl and 2.5 wt % urea in water, to which 12 Nhydrochloric acid had been added to achieve a pH of about 5.

As for Examples 1-4, samples 14-25 were tested for efficacy against C.difficile spores, according to the ASTM E2783-11: “Standard Test Methodfor Assessment of Antimicrobial Activity for Water Miscible CompoundsUsing a Time-Kill Procedure.” Contact time was 30 seconds. Temperaturewas room temperature. Results are shown in Table 3 and FIG. 4.

TABLE 3 Log₁₀ Reduction C. difficile Example Composition pH spores 1480% ethanol 1.5 −0.01 15 80% ethanol + 2% aminoguanidine-HC1 1.5 0.98 1680% ethanol + 2.5% aminoguanidine- 1.5 2.38 HC1 + 2.5% 17 90% ethanol1.5 0.05 18 90% ethanol + 2% aminoguanidine-HCl 1.5 −0.13 19 90%ethanol + 2.5% aminoguanidine- 1.5 3.30 HC1 + 2.5% urea 20 80% ethanol5.0 −0.05 21 80% ethanol + 2% aminoguanidine-HC1 5.0 −0.12 22 80%ethanol + 2.5% aminoguanidine- 5.0 −0.06 HC1 + 2.5% 23 90% ethanol 5.0−0.11 24 90% ethanol + 2% aminoguanidine-HC1 5.0 −0.05 25 90% ethanol +2.5% aminoguanidine- 5.0 −0.07 HC1 + 2.5% urea

Example 26 was a solution of 70 wt. % ethanol in water, to which 12 Nhydrochloric acid had been added to achieve a pH of about 1.5.

Example 27 was a solution of 70 wt. % ethanol, 0.1 wt. % guanidine-HClin water, to which 12 N hydrochloric acid had been added to achieve a pHof about 1.5.

Example 28 was a solution of 70 wt. % ethanol and 1 wt. % guanidine-HClin water, to which 12 N hydrochloric acid had been added to achieve a pHof about 1.5.

Example 29 was a solution of 70 wt. % ethanol and 10 wt. % guanidine-HClin water, to which 12 N hydrochloric acid had been added to achieve a pHof about 1.5.

As for Examples 1-4, samples 26-29 were tested for efficacy against C.difficile spores, according to the ASTM E2783-11: “Standard Test Methodfor Assessment of Antimicrobial Activity for Water Miscible CompoundsUsing a Time-Kill Procedure.” Contact time was 30 seconds. Temperaturewas room temperature. Results are shown in Table 4 below.

TABLE 4 Log₁₀ Reduction Example Composition pH C. difficile spores 2670% ethanol 1.5 0.51 27 70% ethanol + 0.1% guanidine 1.5 0.84 28 70%ethanol + 1.0% guanidine 1.5 2.00 29 70% ethanol + 10.0% guanidine 1.5>3.08

Example 30 was a simple hand wash.

Example 31 was a solution of 70 wt. % ethanol in water, to which 12 Nhydrochloric acid had been added to achieve a pH of about 1.5.

Example 32 was a solution of 70 wt. % ethanol and 10 wt. % urea inwater, to which 12 N hydrochloric acid had been added to achieve a pH ofabout 1.5.

The samples were evaluated at room temperature for efficacy against C.difficile spores according to the following In vivo test protocol.Results are shown in Table 5 and FIG. 5.

In vivo C. difficile Spore Test Method Protocol:

Spore Growth Media and Neutralizer

-   -   BPB+: Butterfield's Phosphate Buffer    -   BHIT-AMP: Brain Heart Infusion Agar with Sodium Taurocholate        hydrate (0.1%) and Ampicillin (0.2 μg/ml)        Inoculum    -   Purified C. difficile spores (ATCC 700057) at an approximate        concentration of 8.5 log₁₀ CFU/ml. Spores were suspended in        sterile water and stored at −80° C.        Pretreatment of Hands    -   Pre-wash hands with bland soap (GOJO® Clear & Mild Foam        Handwash) and pat dry with paper towels and wait five minutes        before applying spore inoculum to hands        Inoculation of Fingertips with C. difficile Spores    -   Dispense 5 μL of the spore suspension directly onto the index,        middle, and ring fingerpads of each hand    -   Rub opposite fingerpads together on and off until dry    -   Wait at least 1 minute before determining the baseline        contamination of the fingers        Baseline Recovery    -   Sample one finger on each hand separately in standard size petri        dishes (100×15 mm) containing 5 mL BPB+ by rubbing for one        minute    -   Pat fingerpads dry on paper towels to remove excess BPB+        Product Application    -   For Handwash: briefly wet hands, apply product (2 pumps of        foaming hand washes), lather for 60 seconds, rinse off excess        lather for 10 seconds, lightly pat dry    -   For disinfectant compositions: (Examples): rub fingertips        together under a continuous stream from a wash bottle filled        with the appropriate product for the desired exposure time,        briefly rinse to neutralize, lightly pat dry        Post Product Exposure Recovery    -   Sample index, middle, and ring fingers of each hand together as        described above        Enumeration of C. difficile    -   Dilute baseline and post product exposure recovery in BPB+ and        enumerate on BHIT-AMP.        Log Reduction Calculation    -   After enumeration of viable C. difficile with subtract post        exposure recovery from baselines to get a log reduction

TABLE 5 Application Log₁₀ Reduction Example Composition pH Time (s) C.difficile spores 30 bland handwash — 60 1.40 31 70% ethanol 2.0 30 1.2532 70% ethanol + 10% urea 1.5 30 4.39

Example 33 was a solution of 70 wt. % ethanol in water.

Example 34 was a solution of 70 wt. % ethanol and 10 wt. % urea inwater, to which 12 N hydrochloric acid had been added to achieve a pH ofabout 1.5.

Example 35 was a solution of 70 wt. % ethanol and 1 wt. % urea in water,to which 12 N hydrochloric acid had been added to achieve a pH of about1.5.

Example 36 was a solution of 70 wt. % ethanol and 10 wt. % urea inwater, to which 12 N hydrochloric acid had been added to achieve a pH ofabout 3.

The samples were evaluated for efficacy against C. difficile sporesaccording to the following in vivo test protocol.

In vivo C. difficile Spore Test Method Protocol:

Spore Growth Media and Neutralizer

-   -   BPB+: Butterfield's Phosphate Buffer    -   BHIT-AMP: Brain Heart Infusion Agar with Sodium Taurocholate        hydrate (0.1%) and Ampicillin (0.2 μg/ml)        Inoculum    -   Purified C. difficile spores (ATCC 700057) at an approximate        concentration of 8.5 log₁₀ CFU/ml. Spores were suspended in        sterile water and stored at −80° C.        Pretreatment of Hands    -   Pre-wash hands with bland soap (GOJO® Clear & Mild Foam        Handwash) and pat dry with paper towels and wait five minutes        before applying spore inoculum to hands        Inoculation of Fingertips with C. difficile spores    -   Dispense 5 μL of the spore suspension directly onto the index,        middle, and ring fingerpads of each hand    -   Rub opposite fingerpads together on and off until dry    -   Wait at least 1 minute before determining the baseline        contamination of the fingers        Baseline Recovery    -   Sample one finger on each hand separately in standard size petri        dishes (100×15 mm) containing 5 mL BPB+ by rubbing for one        minute    -   Pat fingerpads dry on paper towels to remove excess BPB+        Product Application    -   Briefly wet hands, apply product (approximately 3 ml), rub hands        together to spread product over hands, and continue rubbing        until product has evaporated.        Post Product Exposure Recovery    -   Sample index, middle, and ring fingers of each hand together as        described above        Enumeration of C. difficile    -   Dilute baseline and post product exposure recovery in BPB+ and        enumerate on BHIT-AMP.        Log Reduction Calculation    -   After enumeration of viable C. difficile with subtract post        exposure recovery from baselines to get a log reduction. Results        are summarized in the table below.

TABLE 6 Application Log₁₀ Reduction Example Composition pH Volume (ml)C. difficile spores 33 70% ethanol — 3.00 0.24 34 70% ethanol + 10% urea1.5 3.00 3.58 35 70% ethanol + 1.0% urea 1.5 3.00 3.87 36 70% ethanol +10% urea 3.0 3.00 1.00

Example 37 was a solution of 70 wt. % isopropanol and 1 wt. % urea inwater, to which 12 N hydrochloric acid had been added to achieve a pH ofabout 1.5.

Example 38 was a solution of 70 wt. % isopropanol and 10 wt. % urea inwater, to which 12 N hydrochloric acid had been added to achieve a pH ofabout 1.5.

Example 39 was a solution of 70 wt. % isopropanol and 1 wt. % urea inwater, to which 12 N hydrochloric acid had been added to achieve a pH ofabout 1.5.

Example 40 was a solution of 70 wt. % isopropanol and 10 wt. % urea inwater, to which 12 N hydrochloric acid had been added to achieve a pH ofabout 1.5.

Example 41 was a solution of 70 wt. % isopropanol and 1 wt. % urea inwater, to which 12 N hydrochloric acid had been added to achieve a pH ofabout 5.

Example 42 was a solution of 70 wt. % isopropanol and 10 wt. % urea inwater, to which 12 N hydrochloric acid had been added to achieve a pH ofabout 5.

Example 43 was a solution of 70 wt. % isopropanol and 1 wt. % urea inwater, to which 12 N hydrochloric acid had been added to achieve a pH ofabout 5.

Example 44 was a solution of 70 wt. % isopropanol and 10 wt. % urea inwater, to which 12 N hydrochloric acid had been added to achieve a pH ofabout 5.

Example 45 was a solution of 70 wt. % n-propanol and 1 wt. % urea inwater, to which 12 N hydrochloric acid had been added to achieve a pH ofabout 1.5.

Example 46 was a solution of 70 wt. % n-propanol and 10 wt. % urea inwater, to which 12 N hydrochloric acid had been added to achieve a pH ofabout 1.5.

Example 47 was a solution of 70 wt. % n-propanol and 1 wt. % urea inwater, to which 12 N hydrochloric acid had been added to achieve a pH ofabout 1.5.

Example 48 was a solution of 70 wt. % n-propanol and 10 wt. % urea inwater, to which 12 N hydrochloric acid had been added to achieve a pH ofabout 1.5.

Example 49 was a solution of 70 wt. % n-propanol and 1 wt. % urea inwater, to which 12 N hydrochloric acid had been added to achieve a pH ofabout 5.

Example 50 was a solution of 70 wt. % n-propanol and 10 wt. % urea inwater, to which 12 N hydrochloric acid had been added to achieve a pH ofabout 5.

Example 51 was a solution of 70 wt. % n-propanol and 1 wt. % urea inwater, to which 12 N hydrochloric acid had been added to achieve a pH ofabout 5.

Example 52 was a solution of 70 wt. % n-propanol and 10 wt. % urea inwater, to which 12 N hydrochloric acid had been added to achieve a pH ofabout 5.

As for Examples 1-4, samples 37-52 were tested for efficacy against C.difficile spores, according to the ASTM E2783-11: “Standard Test Methodfor Assessment of Antimicrobial Activity for Water Miscible CompoundsUsing a Time-Kill Procedure.” Contact time was 30 seconds or 60 seconds,as indicated in the graph below. Temperature was room temperature.Results are shown in the table below.

TABLE 7 Exposure Log₁₀ Reduction Example Composition pH time C.difficile spores 37 70% isopropanol + 1% urea 1.5 30 −0.77 38 70%isopropanol + 10% urea 1.5 30 3.31 39 70% isopropanol + 1% urea 1.5 60−0.33 40 70% isopropanol + 10% urea 1.5 60 >3.46 41 70% isopropanol + 1%urea 5.0 30 −0.37 42 70% isopropanol + 10% urea 5.0 30 −0.38 43 70%isopropanol + 1% urea 5.0 60 −0.38 44 70% isopropanol + 10% urea 5.0 60−0.14 45 70% n-propanol + 1% urea 1.5 30 0.26 46 70% n-propanol + 10%urea 1.5 30 >3.46 47 70% n-propanol + 1% urea 1.5 60 0.67 48 70%n-propanol + 10% urea 1.5 60 3.31 49 70% n-propanol + 1% urea 5.0 30−0.06 50 70% n-propanol + 10% urea 5.0 30 0.18 51 70% n-propanol + 1%urea 5.0 60 0.10 52 70% n-propanol + 10% urea 5.0 60 0.18

Example 53 was a solution of 70 wt. % ethanol and 1 wt. % thiourea inwater, to which 12 N hydrochloric acid had been added to achieve a pH ofabout 1.5.

Example 54 was a solution of 70 wt. % ethanol and 10 wt. % thiourea inwater, to which 12 N hydrochloric acid had been added to achieve a pH ofabout 1.5.

Example 55 was a solution of 70 wt. % ethanol and 1 wt. % guanidinethiocyanate in water, to which 12 N hydrochloric acid had been added toachieve a pH of about 1.5.

Example 56 was a solution of 70 wt. % ethanol and 10 wt. % guanidinethiocyanate in water, to which 12 N hydrochloric acid had been added toachieve a pH of about 1.5.

Example 57 was a solution of 70 wt. % ethanol and 1 wt. %L-N^(G)-Nitroarginine in water, to which 12 N hydrochloric acid had beenadded to achieve a pH of about 1.5.

As for Examples 1-4, samples 53-57 were tested for efficacy against C.difficile spores, according to the ASTM E2783-11: “Standard Test Methodfor Assessment of Antimicrobial Activity for Water Miscible CompoundsUsing a Time-Kill Procedure.” Contact time was 30 seconds. Temperaturewas room temperature. Results are shown in the table below.

TABLE 8 Log₁₀ Reduction Example Composition pH C. difficile spores 5370% ethanol + 1% thiourea 1.5 >2.90 54 70% ethanol + 10% thiourea 1.52.75 55 70% ethanol + 1% guanidine thiocyanate 1.5 >2.90 56 70%ethanol + 10% guanidine thiocyanate 1.5 2.51 57 70% ethanol + 1%L-NG-Nitroarginine* 1.5 >2.90 *CAS # for this molecule: 2149-70-4

Example 58 was a solution of 70 wt. % ethanol in water, to which 12 Nhydrochloric acid had been added to achieve a pH of about 3.

Example 59 was a solution of 70 wt. % ethanol and 10 wt. % urea inwater, to which 12 N hydrochloric acid had been added to achieve a pH ofabout 3.

Example 60 was a solution of 70 wt. % ethanol in water, to which 12 Nhydrochloric acid had been added to achieve a pH of about 7.

Example 61 was a solution of 70 wt. % ethanol and 10 wt. % urea inwater, to which 12 N hydrochloric acid had been added to achieve a pH ofabout 7.

As for Examples 1-4, samples 58-61 were tested for efficacy against C.difficile spores, according to the ASTM E2783-11: “Standard Test Methodfor Assessment of Antimicrobial Activity for Water Miscible CompoundsUsing a Time-Kill Procedure.” Contact time was 5 minutes. Temperaturewas room temperature. Results are shown in the table below.

TABLE 9 Exposure Log₁₀ Reduction Example Composition pH time (min.) C.difficile spores 58 70% ethanol 3 5 0.04 59 70% ethanol + 10% urea 3 53.14 60 70% ethanol 7 5 −0.35 61 70% ethanol + 10% urea 7 5 0.10

Example 62 was a solution of 70 wt. % ethanol in water, to which 12 Nhydrochloric acid had been added to achieve a pH of about 2.

Example 63 was a solution of 70 wt. % ethanol, 10 wt. % urea and 1%NaNO₂ in water, to which 12 N hydrochloric acid had been added toachieve a pH of about 2.

Example 64 was a solution of 80 wt. % ethanol in water, to which 12 Nhydrochloric acid had been added to achieve a pH of about 3.

Example 65 was a solution of 70 wt. % ethanol, 10 wt. % urea, and 1%NaNO₂ in water, to which 12 N hydrochloric acid had been added toachieve a pH of about 3.

Example 66 was a solution of 70 wt. % ethanol, 10 wt. % urea, and 1 wt.% tannic acid in water, to which 12 N hydrochloric acid had been addedto achieve a pH of about 2.

Example 67 was a solution of 70 wt. % ethanol, 10 wt. % urea, and 1 wt.% citric acid in water, to which 12 N hydrochloric acid had been addedto achieve a pH of about 2.

Example 68 was a solution of 70 wt. % ethanol, 10 wt. % urea, and 1 wt.% lauric acid in water, to which 12 N hydrochloric acid had been addedto achieve a pH of about 2.

Example 69 was a solution of 70 wt. % ethanol, 10 wt. % urea, and 0.1wt. % sodium dodecyl sulfate (SDS) in water, to which 12 N hydrochloricacid had been added to achieve a pH of about 3.

Example 70 was a solution of 70 wt. % ethanol, 10 wt. % urea, and 1 wt.% SDS in water, to which 12 N hydrochloric acid had been added toachieve a pH of about 3.

Example 71 was a solution of 70 wt. % ethanol and 1 wt. % sodium laurethsulfate (SLES) in water, to which 12 N hydrochloric acid had been addedto achieve a pH of about 1.5.

Example 72 was a solution of 70 wt. % ethanol and 5 wt. % sodium laurylsulfate (SLS) in water, to which 12 N hydrochloric acid had been addedto achieve a pH of about 3.

As for Examples 1-4, samples 62-72 were tested for efficacy against C.difficile spores, according to the ASTM E2783-11: “Standard Test Methodfor Assessment of Antimicrobial Activity for Water Miscible CompoundsUsing a Time-Kill Procedure.” Temperature was room temperature. Resultsare shown in the table below.

TABLE 10 Log₁₀ Exposure Reduction time C. difficile Example CompositionpH (sec) spores 62 70% ethanol 2 30 −0.13 63 70% ethanol + 10% urea + 230 >3.0 1% NaNO2 64 80% ethanol 3 30 −0.09 65 70% ethanol + 10% urea + 330 >3.0 1% NaNO2 66 70% ethanol + 10% urea + 1% tannic 2 30 3.29 67 70%ethanol + 10% urea + 1% citric 2 30 3.13 68 70% ethanol + 10% urea + 1%lauric 2 30 3.13 69 70% ethanol + 10% urea + 0.1% SDS 3 30 2.44 70 70%ethanol + 10% urea + 1% SDS 3 30 >3.13 71 70% ethanol + 1% SLES 1.560 >2.9 72 70% ethanol + 5% SLS 1.5 30 1.87

Example 73 was a solution of 70 wt. % ethanol in water, to which 12 Nhydrochloric acid had been added to achieve a pH of about 1.5.

Example 74 was a solution of 70 wt. % ethanol and 0.25 wt. % NaCl inwater, to which 12 N hydrochloric acid had been added to achieve a pH ofabout 1.5.

Example 75 was a solution of 70 wt. % ethanol and 0.1 wt. % Trilon M inwater, to which 12 N hydrochloric acid had been added to achieve a pH ofabout 1.5.

Example 76 was a solution of 70 wt. % ethanol and 0.5 wt. % Trilon M inwater, to which 12 N hydrochloric acid had been added to achieve a pH ofabout 1.5.

Example 77 was a solution of 70 wt. % ethanol and 1 wt. % Trilon M inwater, to which 12 N hydrochloric acid had been added to achieve a pH ofabout 1.5.

Example 78 was a solution of 70 wt. % ethanol and 2 wt. % Trilon M inwater, to which 12 N hydrochloric acid had been added to achieve a pH ofabout 1.5.

Example 79 was a solution of 70 wt. % ethanol, 0.25 wt. % NaCl, and 1wt. % Trilon M in water, to which 12 N hydrochloric acid had been addedto achieve a pH of about 1.5.

Example 80 was a solution of 70 wt. % ethanol, 0.25 wt. % NaCl, and 2wt. % Trilon M in water, to which 12 N hydrochloric acid had been addedto achieve a pH of about 1.5.

As for Examples 1-4, samples 73-80 were tested for efficacy against C.difficile spores, according to the ASTM E2783-11: “Standard Test Methodfor Assessment of Antimicrobial Activity for Water Miscible CompoundsUsing a Time-Kill Procedure.” Temperature was room temperature. Resultsare shown in the table below.

TABLE 11 Exposure Log₁₀ Reduction Example Composition pH Time (s) C.difficile spores 73 70% ethanol 1.50 30 0.26 74 70% ethanol + 0.25% NaCl1.50 30 1.92 75 70% ethanol + 0.1% Trilon M 1.50 30 1.35 76 70%ethanol + 0.5% Trilon M 1.50 30 2.02 77 70% ethanol + 1.0% Trilon M 1.5030 3.31 78 70% ethanol + 2.0% Trilon M 1.50 30 >3.46 79 70% ethanol +0.25% NaCl + 1.0% Trilon M 1.50 30 >3.46 80 70% ethanol + 0.25% NaCl +2.0% Trilon M 1.50 30 >3.46

Example 81 was a solution of 70 wt. % ethanol and 1 wt. % NaCl in water,to which 12 N hydrochloric acid had been added to achieve a pH of about1.5.

Examples 3, 10, 72 and 81 were tested for efficacy against C. difficilespores according to the EPA Standard Operating Procedure forQuantitative Disk Carrier Test method (QCT-2) Modified for TestingAntimicrobial Products Against Spores of Clostridium difficile (ATCC43598) on Inanimate, Hard, Non-porous Surfaces.

SOP Number: MB-31-03. Date Revised Jun. 12, 2014, which is incorporatedby reference herein. This quantitative method is used to evaluate thesporicidal efficacy of liquid disinfectants against spores ofClostridium difficile (ATCC 43598) on inanimate, hard, non-poroussurfaces. Results are shown in the table below.

TABLE 12 Log Reduction Log Reduction C. difficile spores C. difficilespores Example pH At 7 minutes At 10 minutes 3 1.50 6.3 6.2 10 1.50 6.26.2 72 1.50 6.20 6.20 81 1.50 6.20 6.20

Various modifications and alterations that do not depart from the scopeand spirit of this invention will become apparent to those skilled inthe art. This invention is not to be duly limited to the illustrativeembodiments set forth herein.

What is claimed is:
 1. A composition for the disinfection of surfaces,the composition comprising: at least 40 wt. % of a C₁₋₆ alcohol, basedupon the total weight of the composition; and at least one primaryenhancer, wherein the composition has a pH of less than
 3. 2. Thecomposition of claim 1, wherein the at least one primary enhancer isselected from the group consisting of amine-containing enhancers,α-aminoacids, salts of alkali metals, salts of alkaline earth metals,and anionic surfactants.
 3. The composition of claim 1, wherein the atleast one primary enhancer is selected from the group consisting ofammonium chloride, ammonium iron citrate, calcium chloride, ironperchlorate, lithium perchlorate, lithium acetate, magnesium chloride,sodium chlorate, sodium chloride, sodium chlorite, 2-amino-2-hydroxymethyl-propane-1,3-diol hydrochloride, and combinations thereof.
 4. Thecomposition of claim 3, wherein the at least one primary enhancer isammonium chloride.
 5. The composition of claim 1, wherein thecomposition exhibits an enhanced efficacy against C. difficile spores,when compared to the efficacy of the alcohol or primary enhancer alone.6. The composition of claim 1, further comprising at least one secondaryenhancer.
 7. The composition of claim 6, wherein the at least onesecondary enhancer is selected from the group consisting of non-ionicsurfactants, organic acids, antimicrobial agents, oxidizing agents,sugars, sugar alcohols, monosaccharides, glycerol and combinationsthereof.
 8. The composition of claim 6, wherein the at least onesecondary enhancer is selected from the group consisting of decylglucoside, polyalkoxylated dimethicones, glycerol, and combinationsthereof.
 9. The composition of claim 7, wherein the organic acid isselected from the group consisting of citric acid, lauric acid, tannicacid, and combinations thereof.
 10. The composition of claim 7, whereinthe antimicrobial agent is selected from the group consisting oftriclosan, PCMX, chloroxylenol, hexetidine, chlorhexidine salts2-bromo-2-nitropropane-1, 3-diol, benzalkonium chloride, cetylpyridiniumchloride, alkylbenzyldimethylammonium chlorides, iodine, phenol,bisphenol, diphenyl ether, phenol derivatives, povidone-iodine,parabens, hydantoins and derivatives thereof, phenoxyethanol, cis isomerof 1-(3-chloroallyl)-3,5,6-triaza-1-azoniaadamantane chloride,diazolidinyl urea, benzethonium chloride, methylbenzethonium chloride,glyceryl laurate, transition metal compounds, hydrogen peroxide,chlorine dioxide, anilides, bisguanidines, tropolone, C₆₋₁₀-alkanediols, and combinations thereof.
 11. A composition for the inactivationof C. difficile spores on surfaces, the composition comprising: at least40 wt. % of a C₁₋₆ alcohol, based upon the total weight of thecomposition; and at least one primary enhancer selected from the groupconsisting of amine-containing enhancers, α-aminoacids, salts of alkalimetals, salts of alkaline earth metals, and anionic surfactants, whereinthe composition has a pH of less than
 3. 12. The composition of claim11, wherein the at least one primary enhancer is selected from the groupconsisting of ammonium chloride, ammonium iron citrate, calciumchloride, iron perchlorate, lithium perchlorate, lithium acetate,magnesium chloride, sodium chlorate, sodium chloride, sodium chlorite,2-amino-2-hydroxy methyl-propane-1,3-diol hydrochloride, andcombinations thereof.
 13. The composition of claim 12, wherein the atleast one primary enhancer is ammonium chloride.
 14. The composition ofclaim 11, further comprising at least one secondary enhancer selectedfrom the group consisting of non-ionic surfactants, organic acids,antimicrobial agents, oxidizing agents, sugars, sugar alcohols,monosaccharides, glycerol and combinations thereof.
 15. A compositionfor the disinfection of surfaces, the composition comprising: at least70 wt. % of a C₁₋₆ alcohol, based upon the total weight of thecomposition; and at least one primary enhancer selected from the groupconsisting of amine-containing enhancers, α-aminoacids, salts of alkalimetals, salts of alkaline earth metals, and anionic surfactants, whereinthe composition has pH of less than
 3. 16. The composition of claim 1,wherein the composition comprises from 0.1 to 20 wt. % of the at leastone primary enhancer, based upon the total weight of the composition.17. The composition of claim 1, wherein the composition has a pH of lessthan 2.75.
 18. The composition of claim 1, wherein the composition has apH of less than
 2. 19. The composition of claim 11, wherein thecomposition has a pH of less than 2.75.
 20. The composition of claim 11,wherein the composition has a pH of less than 2.